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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Three in vitro tests (Ames, gene mutation and chromosome aberration assays in mammalian cells) were performed and were negative. The conclusion on gene mutation is therefore “Not classified - based on specific, valid data on the substance”.
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998/04/20 to 1998/05/22
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Mutation in Histidine biosynthesis for Salmonella typhimurium: his D 3052/strain TA98 (frameshift), his G 46/strains TA100 and TA1535 (base-pair
substitution) and his C 3076/strain TA1537 (frameshift)
Mutation in Tryptophan biosynthesis for Escherichia coli : WP2uvrA- (deletion in an excision repair gene)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
50 - 150 - 500 - 1500 and 5000 µg/plate
Vehicle / solvent:
dimethyl formamide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylformamide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
for TA98 strain without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylformamide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
for TA100, TA1535, WP2uvrA without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylformamide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA1537 without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylformamide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA100, TA1535, TA1537 and WP2uvrA with S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethylformamide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
for TA98 with S9 activation
Details on test system and experimental conditions:
(1) Preliminary evaluation of cytotoxicity of the substance
The cytotoxicity was performed of the Salmonella Typhimurium strain TA 100 and in the Escherichia Coli WP2uvrA- with and without metabolic activation (S9 mix). The substance was tested at 10 concentrations : 0.15 ; 0.5 ; 1.5 ; 5 ; 15; 50; 150 ; 500 ; 1500 and 5000 µg/dish. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was
plated onto a sterile plates of Vogel-Bonner Minimal agar. The Petri dishes were incubated at 37°C for 48 hours.
(2) Main study
4 strains of Salmonella Typhimurium were tested: TA98, TA100, TA1535 and TA1537. 1 strain of Escherichia Coli was tested : WP2uvrA-
As the concentration of 5 mg/plate was not toxic in the strain TA 100 and UWP2uvrA- with and without metabolic activation during the preliminary
study, the following concentrations were tested during the main study: 50 ; 150 ; 500; 1500 and 5000 µg/dish.
Each concentration was tested 3 times under a constant volume of 0.1 ml with and without metabolic activation (S9 fraction obtained from rat livers pre-treated with Aroclor 1254 at 500 mg/kg) and used at on each strain. The incubation system contained 0.1 ml of the diluted substance + 2 ml of
top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was plated onto the surface of Vogel-Bonner Minimal agar plate and incubated at 37°C for 48 hours.
Dimethyl formamide and positive controls, 4-Nitroquinoline-1-oxide 0.2 µg/plate for TA-98; N-ethyl-N'-nitro-nitroguanidine at 3 µg/plate for
TA-100, at 5 µg/plate for TA-1535 and at 2 µg/plate for WP2uvrA-; 9-aminoacridine at 80 µg/plate for TA-1537. 2-aminoanthracene was used as positive reference, in presence of S9 mix, at 1 µg/plate for TA100 ; at 2 µg/plate for TA1535 and TA1537 and at 10 µg/plate for WP2uvrA-.
Benzo(a)pyrene was used, in presence of S9 mix, at 5 µg/plate for TA98.
All the results were confirmed in a second study independent from the first.
Evaluation criteria:
(1) Preliminary cytotoxicity
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of bacterial background lawn. Manual counts were performed ate and above 1500 µg/plate because of test material precipitation.
(2) Reverse mutation
At the end of the incubation period, the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at and above 1500 µg/plate because of test material precipitation.
The test material may considered to be positive in the test system if it should have induced a reproducible, dose-related and statistically significant
increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments
then this is taken as evidence of a positive response.
Statistics:
Dunnett's method of linear regression
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxicity was eshibited to any of the strains of bacteria used. A white, flaky particulate precipitate was observed at and above 1500 µg/plate.
Under experimental conditions employed, no value obtained in the presence of the test article was greater than or equal to twice the value obtained in the presence of the vehicle with and without metabolic activation on the bacterial strains used.
All the results obtained in presence of positive controls were significant with and without metabolic activation in the used bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test article MONTANOV 202, is not mutagenic as it induces no significant increase in the number of revertants with and without metabolic
activation of Salmonella Typhimurium TA98, TA100, TA1535 and TA1537 and in Escherichia Coli WP2uvrA-.
Executive summary:

The substance MONTANOV 202 was tested on 4 strains of salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in 1 strain of Escherichia Coli (WP2uvrA-), with or without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strains TA-100 and WP2uvrA- with and without metabolic activation.

The 5 concentrations (50 - 150 - 500 - 1500 and 5000 µg/plate) were tested 3 times on the 5 strains mentioned above with and without metabolic activation. The results were confirmed in a second study, independant of the first.

In each study was included a negative control (vehicle = dimethyl formamide) and a positive control (specific standard mutagen).

Under the experimental conditions employed, the substance Montanov 202 did not show any mutagenic potential for the strain TA98, TA100, TA1535, TA1537 and WP2uvrA- with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008/04/07 to 2008/05/23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2005-25-08
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
numerical and structural aberrations for chromosome or chromatid.
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
human peripheral blood lymphocytes obtained from peripheral blood of a healthy adult male.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
(1) solubility assay
According to the solubility of the test substance, the higher concentration tested was 500 µg/ml in heated sterile millipore water (80°C).
(2) Cytotoxicity
According to the solubility assay, 5 concentrations were tested for the cytotoxic assay: 31.25 - 62.5 - 125 - 250 and 500 µg/ml.
(3) Main study
According to the cytotoxicity assay, 5 concentrations were tested: 204.8 - 256 - 320 - 400 and 500 µg/ml.
Vehicle / solvent:
Sterile millipore water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile millipore water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile millipore water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 activation
Details on test system and experimental conditions:
The assay was performed in cultured human peripheral blood lymphocytes obtained from peripheral blood of a healthy adult male.
A range finding study was first performed in a preliminary study to assess the cytotoxicity based on mitotic index and microscopic evaluation. This
preliminary study fixed the high dose for the main study.
According to the cytotoxic preliminary assay, the concentrations chosen for the main study were 204.8 ; 256 ; 320 ; 400 and 500 µg/ml
with (10% S9) and without S9. Vehicle (1% sterile milipore water) and positive controls were tested in parallel. The solvent control received 1 % sterile millipore water. Positive controls employed for with and without S9 were cyclophosphamide and mitomycin C respectively, at concentrations 20 and 1 µg/ml respectively.
The cultures were tested in duplicates for all the treatment conditions.
The PHA-M stimulated cultures were maintained in a humidified atmosphere of 5% CO2 at 37 ± 0.5 °C. After the test substance addition at the 48th
hour with and without S9, cultures were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times corresponding to 36
hours. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical -colchicine at a concentration of 0.4 μg/ml.
Metaphase preparations were made from cells in fixative, after hypotonic shock with pre incubated 0.075M KCl at 37 °C and a series of washes with
fixative (methanol: acetic acid at 3:1) at 1600 rpm for 10 min. Heat fixed preparations were stained with Giemsa. Thousand consecutive cells were
scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases with 46 ± 2
centromeres were analyzed for aberrations. The observations were recorded and summarized as percentage numerical aberrations and mean
structural aberrations.

Evaluation criteria:
Evaluation of cytotoxicity of the substance according to the mitotic index and microscopic observation.
For each condition of the main study, the following parameters were studied :
- number of cells scored
- percentage mitotic indices
- frequencies of structural and numerical aberrations
- percentage of cells with aberration (excluding gaps): polyploidy, fragment, deletion, pulverized chromosome, endoreduplication, break, exchange, dicentric, ring
Statistics:

comparison between the various treatment groups according to the Student's T test (p<0.05)
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary study performed to evaluation the cytotoxicity of the substance, three concentrations were first employed 31.25 - 62.5 - 125 - 250 - 500 µg/ml, with (10% S9) and without metabolic activation system (S9). No cytotoxicity was observed at all concentrations.
From the results of the range finding studies, 500 µg/ml was chosen as the high dose for the main study.
In the main study with 4 hours exposure time period and with continous exposure period, the % mitotic indices of the concentrations tested (with
and without S9), 204.8 - 256 - 320 - 400 and 500 µg/ml were comparable with the solvent control. Corresponding values were obtained in the
duplicate.
At 204.8 - 256 - 320 - 400 and 500 µg/ml in the 4h exposure time period and the continuous exposure time period, with and without S9, a lack of induction of chromosome aberrations was observed. It was in consensus with the solvent control. The frequencies of polyploidy was insignificant
amongst the cultures treated with the substance and the vehicle in both the exposure periods and with and without S9.
The cultures treated with known mutagens in the 4h exposure time period and the continuous exposure time period, with and without S9, exhibited a statistically significant increase in the mean frequency of structural aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: human peripheral blood lymphocytes obtained from peripheral blood of a healthy adult male.
Conclusions:
Under the test conditions employed, the substance LCE07104 did not induce any apparent numerical and structural aberrations both in presence
and absence of an exogenous mammalian metabolic activation component (S9) in the 4h exposure and continuous exposure time periods. Hence,
the substance LCE07104 may be considered non genotoxic in the in vitro chromosome aberrations assay on human lymphocytes.
Executive summary:

The ability of the substance LCE07104 to induce numerical and structural cytogenetic anomalies was evaluated by enumerating the incidence of chromosome aberrations in cultured human peripheral blood lymphocytes.

Two range finding studies were performed to assess the cytotoxicity based on mitotic index, to fix the high dose for the main study. The following concentrations of the test substance were employed, 31.25, 62.50, 125, 250 and 500 µg/ml with (10% S9) and without metabolic activation system (S9). No cytotoxicity was observed at all concentrations with and without S9. From the results of the range finding studies, 500 µg/ml was chosen as the high dose for the main study.The concentrations chosen for the main study were 204.8 - 256 - 320 - 400 and 500 µg/ml with (10% S9) and without S9. Concurrent solvent (1% sterile millipore water) and positive controls were tested. Positive controls employed for with and without S9 were cyclophosphamide and mitomycin C respectively, at concentrations 20 and 1 µg/ml respectively. The cultures were maintained in duplicates for all the treatment conditions.

The test substance, vehicle and positive controls were allowed for an exposure period of 4 h and an expression period of 1.5 cell cycle times. One hour prior to harvest, the cultures were treated with a metaphase arresting chemical.

Thousand consecutive cells were scored and cells in metaphase were enumerated for deriving the percentage mitotic index. Two hundred well spread intact metaphases with 46 ± 2 centromeres were analyzed for aberrations. The observations were

recorded and summarized as percentage numerical aberrations and mean structural aberrations.

Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested with and without metabolic activation in the 4 h exposure time period. Cultures treated with the test substance exhibited statistically comparable values of numerical and structural aberrations to that of the solvent control group with and without

S9. However, the positive control groups exhibited statistically increased frequencies of cytogenetic anomalies thus validating the sensitivity of the assay.

Based on the negative results from the 4 h exposure time period, an additional experiment was performed with continuous exposure in the presence of the test substance for 1.5 cell cycle time without S9. The concentrations employed were 204.8 - 256 - 320 - 400 and 500 μg/ml and the experiment was performed in a similar pattern as detailed in the 4 h exposure time period. Under the test conditions employed, no apparent induction of numerical or structural aberrations was observed in any of the concentrations tested without metabolic activation in the continuous exposure time period. Cultures treated with the test substance exhibited statistically comparable values of numerical and structural aberrations to that of the solvent control group. However, the

positive control group exhibited statistically increased frequencies of cytogenetic anomalies thus validating the sensitivity of the assay.

Thus, the substance LCE07104 was considered non genotoxic in the in vitro chromosome aberration assay on human lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-28 to 2008-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (tk) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
(1) solubility assay
According to the solubility of the test substance, the higher concentration tested was 250 µg/ml in heated sterile millipore water (80°C).
(2) Cytotoxicity
According to the solubility assay, 9 concentrations were tested for the cytotoxic assay: 41.97 ; 52.46 ; 65.57 ; 81.92 ; 102.4 ; 128 ; 160 ; 200 and 250 µg/ml.
(3) Main study
According to the cytotoxicity assay, 5 concentrations were tested: 15.63 ; 31.25 ; 62.5 ; 125 and 250 µg/ml.
Vehicle / solvent:
sterile millipore water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile millipore water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile millipore water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 activation
Details on test system and experimental conditions:
(1) evaluation of cytotoxiciy (preliminary study)
L5178Y TK +/- cells treated for 3 hours witth sterile water alone (solvent control) or with 9 concentrations of the test substance with or without S9 activation. After washing, cells were subsequently cultured for 24hours. 24 hours and 3 days after recounting and dilution. Viable clones were counted visually using haemocytometer. The relative total growth (RTG) was calculated.
(2) evaluation of gene mutation potential (main study)
Cultures were treated for 3 or 24 hours with test substance, known mutagens (benzo-a-pyrene 2 and 3 µg/ml and methylmethanesulfonate 10 and 20 µg/ml) with or without S9. The solubility of the test substance was assessed visually, at the begining and end of the treatment.
Each assay was performed in duplicate.
After the 3 or 24 hours treatment period, the cells were washed, pelleted and resuspended for 2 days. During this time, the tk- mutation was expressed. At the end of the expression period, cells were incubated for 2 weeks with or without trifluorothymidine (TFT) at 3 µg/ml (final concentration) was added to the cell to establish the TK mutation. The Mutant Frequency (MF) was calculated.
Evaluation criteria:
(1) Cytotoxicity
The highest concentration for the main study was chosen on the basis that it exhibited 10-20% RTG.
(2) Gene mutation
A concentration-related or a reproducible increase in the mutant frequency is amongst the several criteria's considered for determining a positive result.
Statistics:
The number of mutant colonies obtained at different concentrations of the test substance was statistically compared to the solvent control using Student's T test, for significance (p<0.05).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no concentration related and reproducible increase in the mutant frequencies of any of the tested concentrations and also no statistically significant dose-response relationship was observed. However, in the positive controls a statistically significant increase in the mutant frequencies was observed and a clear indication of clastogenic activity was indicated by a proportional increase in both the small and large colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Hence the test substance was considered as non mutagenic in the mouse lymphoma assay, under the given test conditions.
Executive summary:

A study in mouse lymphoma L5178Y TK+/- cell line was performed to measure mutation at thymidine kinase (TK) locus with and without exogenous mammalian (rat liver S9) microsomal metabolic activation in order to evaluate the ability of LCE07104, sponsored by SEPPIC, France, to induce gene mutation. A range finding study was performed with 3 h and 24 h treatments with and without S9. The concentrations chosen were 41.97, 52.46, 65.57, 81.92, 102.4, 128, 160, 200 and 250 µg/ml, and the maximum possible dose being 250 µg/ml according to the precipitate obtained in the culture media. The test substance was dissolved in sterile millipore water heated at 80°C. An emulsion was obtained. Tested at higher concentrations or with other solvent (like DMSO) immiscibility or precipate were obtained . The % Relative Total Growth (RTG) for the above mentioned concentrations were 72.13, 78.01, 58.05, 50.08, 50.58, 52.89, 61.27, 45.19 and 40.20 respectively with S9 and 70.99, 66.46, 51.91, 61.04, 60.55, 48.28, 56.03, 44.05 and 47.16 respectively without S9 in the 3 h treatment. The % Relative Total Growth (RTG) for the above mentioned concentrations in the 24 h treatment without S9 were 73.38, 63.74, 77.74, 56.26, 48.25, 56.16, 56.54, 53.27 and 48.37 respectively. The high dose chosen for the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was 250 µg/ml, as the % RTG was found comparable to that of the solvent control.

Based on the results of the range finding study, the main study - 3 h treatment with and without S9 and 24 h treatment without S9 was performed employing concentrations 15.63, 31.25, 62.5, 125 and 250 µg/ml.

Two positive controls, Methylmethanesulfonate (MMS) in DMSO at concentrations 10 and 20 µg/ml without S9 and Benzo [a] pyrene (BP) at concentrations 2 and 3 µg/ml with S9 were used. The solvent control received 0.1 ml of sterile millipore water alone.

The mutant frequency (MF) in the 3 h treatment (Experiment 1) for concentrations 15.63, 31.25, 62.5, 125 and 250 µg/ml ranged 71.49 to 84.58 with S9 and 66.10 to 79.06 without S9. This was comparable to the solvent control which exhibited MF of 68.85 with S9 and 68.39 without S9. Similarly, the mutant frequency (MF) in the 24 h treatment (Experiment 1) for 15.63, 31.25, 62.5, 125 and 250 µg/ml ranged 70.77 to 86.55 without S9. This was comparable to the solvent control which exhibited MF of 64.74 without S9.

These values were statistically on par with the solvent control. A similar trend was observed in the respective duplicates (Experiment 2) in the 3 h and 24 h treatments.

The positive controls in 3 h treatment (Experiment 1), BP at concentrations 2 and 3 µg/ml exhibited MF 405.98 and 491.30 and MMS at concentrations 10 and 20 µg/ml exhibited MF 372.00 and 558.06. The positive control in 24 h treatment (Experiment 1),

MMS at concentrations 10 and 20 µg/ml exhibited MF 434.05 and 586.47. A statistically significant increase in the small and large colonies and mutant frequencies in the positive mutagen treated cultures confirmed the sensitivity of the assay.

Thus, from the above data no evidence of test substance induced gene mutation in L5178Y TK+/- mouse lymphoma cell line was observed under the given test conditions.

Hence, LCE07104 was considered apparently non-mutagenic in the mouse lymphoma assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo data is available but it is not necessary as all the in vitro data were negative

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The substance is not classified as mutagenic or genotoxic